Diagnosis of 5' and 3' Ds Ends and Ds Cassette Movement

PCR is performed using either a 5' or 3' specific primer combined with
a genomic specific primer to confirm the site of insertion for each
line. Expected sizes are given for each reaction.
Occasionally we will see an additional band or bands, depending on the
reaction conditions and reagents used. We have included these
bands when they appear for reference purposes, but do not explain them
based on the sequence. It is possible that secondary genomic
sites exist producing an alternate product. In addition, the
presence of gene families or tandem copies of genes may complicate the
reactions.

Primers specific to the Ds
and to genomic sequence in PCR reactions are also used to evaluate
movement of the cassette. A hemizygous transposition will
produce both the native amplification product, between the genomic
primers in the non-transgenic Golden Promise line, as well as the TNP
line's diagnostic product from the Ds
end into the genomic sequence. In some cases, the native
amplification product is produced in the TNP lines as well. We
believe such cases to contain additional copies of the gene or sequence
identical to the sequence surrounding the Ds insertion.
All TNP lines have been progeny tested and are homozygous.

PCR to diagnose 5’and 3' Ds
ends are used since the Ac Transposase lines contain the same
selectable marker as the Ds
transposable cassette (Bar). Negative selection for AcTransposase
using PCR must be used after crosses are made to the Ac containing line
to confirm the absence of the transposase in order to maintain a stable
line. The abbreviated Ac Transposase is present without the Ds
ends to disable autonomous movement of the transposase gene.